Project 3 Summary A common strategy employed by Staphylococcus aureus to survive in the host is to develop into an encased community of cells called a biofilm. Our central PPG hypothesis is that S. aureus biofilm architecture and metabolism creates a unique niche that promotes an immune suppressive environment. In this proposal (Project 3), we are focusing on the architecture aspects of this hypothesis by identifying critical components of the biofilm matrix, and in the case of methicillin-resistant S. aureus (MRSA) isolates, this matrix material is predominantly proteins and extracellular DNA (eDNA). We have developed a new approach to identify eDNA- binding proteins that are part of the biofilm matrix. In this proposal, we will characterize these matrix proteins through a series of biofilm, imaging, regulation, and infection model studies. In Specific Aim 1, we will confirm our new method to identify candidate proteins and characterize the biofilm phenotypes of mutants using various assays. We will also assess DNA binding activity and determine whether immunotherapy has an anti- biofilm effect. In Specific Aim 2, we will investigate the regulation of biofilm matrix composition in collaboration with Dr. Bayles (Project 1). We demonstrated that SaeP binds DNA and high level expression causes a dramatic up-regulation in biofilm capacity. We will assess the properties of the SaeP hyper-biofilm and investigate whether the Sae regulatory system senses nucleic acid content in a SaeP dependent manner. Finally, in Specific Aim 3 we will investigate the influence of biofilm matrix proteins on virulence and the establishment of an anti-inflammatory environment during an MRSA orthopedic implant infection model in collaboration with Dr. Kielian (Project 4). We will test eDNA-binding protein mutants in the model, image mutants using nuclease-activatable probes, and assess the effects of mutants on M2 macrophage polarization and establishment of the anti-inflammatory biofilm milieu. Taken together, the proposed studies will advance our understanding of the complex architecture of MRSA biofilms that is important for these infections to persist in the host.